It has recently been demonstrated that mouse and human fibroblasts can be reprogrammed into an embryonic stem cell-like state by introducing combinations of four transcription factors. However, the therapeutic potential of such induced pluripotent stem (iPS) cells remained undefined. By using a humanized sickle cell anemia mouse model, we show that mice can be rescued after transplantation with hematopoietic progenitors obtained in vitro from autologous iPS cells. This was achieved after correction of the human sickle hemoglobin allele by gene-specific targeting. Our results provide proof of principle for using transcription factor-induced reprogramming combined with gene and cell therapy for disease treatment in mice. The problems associated with using retroviruses and oncogenes for reprogramming need to be resolved before iPS cells can be considered for human therapy.

During cellular reprogramming only a small fraction of cells become induced pluripotent stem cells (iPSCs). Previous analyses of gene expression during reprogramming were based on populations of cells, impeding single-cell level identification of reprogramming events. We utilized two gene expression technologies to profile 48 genes in single cells at various stages during the reprogramming process. Analysis of early stages revealed considerable variation in gene expression between cells in contrast to late stages. Expression of Esrrb, Utf1, Lin28, and Dppa2 is a better predictor for cells to progress into iPSCs than expression of Fbxo15, Fgf4, and Oct4 previously suggested to be reprogramming markers. Stochastic gene expression early in reprogramming is followed by a late hierarchical phase with Sox2 being the upstream factor in a gene expression hierarchy. Finally, downstream factors derived from the late phase, which do not include Oct4, Sox2, Klf4, c-Myc and Nanog, can activate the pluripotency circuitry.

SummaryRecent studies have aimed to convert cultured human pluripotent cells to a naive state, but it remains unclear to what extent the resulting cells recapitulate in vivo naive pluripotency. Here we propose a set of molecular criteria for evaluating the naive human pluripotent state by comparing it to the human embryo. We show that transcription of transposable elements provides a sensitive measure of the concordance between pluripotent stem cells and early human development. We also show that induction of the naive state is accompanied by genome-wide DNA hypomethylation, which is reversible except at imprinted genes, and that the X chromosome status resembles that of the human preimplantation embryo. However, we did not see efficient incorporation of naive human cells into mouse embryos. Overall, the different naive conditions we tested showed varied relationships to human embryonic states based on molecular criteria, providing a backdrop for future analysis of naive human pluripotency.

Summary The Tet family of enzymes (Tet1/2/3) converts 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC). Mouse embryonic stem cells (mESCs) highly express Tet1 and have an elevated level of 5hmC. Tet1 has been implicated in ESC maintenance and lineage specification in vitro but its precise function in development is not well defined. To establish the role of Tet1 in pluripotency and development we have generated Tet1 mutant mESCs and mice. Tet1−/− ESCs have reduced levels of 5hmC, subtle changes in global gene expression, are pluripotent and support development of live-born mice in tetraploid complementation assay but display skewed differentiation towards trophectoderm in vitro. Tet1 mutant mice are viable, fertile and grossly normal though some mutant mice have a slightly smaller body size at birth. Our data suggest that Tet1 loss leading to a partial reduction in 5hmC levels does not affect pluripotency in ESCs and is compatible with embryonic and postnatal development.

The study of induced pluripotency is complicated by the need for infection with high-titer retroviral vectors, which results in genetically heterogeneous cell populations. We generated genetically homogeneous ‘secondary’ somatic cells that carry the reprogramming factors as defined doxycycline (dox)-inducible transgenes. These cells were produced by infecting fibroblasts with dox-inducible lentiviruses, reprogramming by dox addition, selecting induced pluripotent stem cells and producing chimeric mice. Cells derived from these chimeras reprogram upon dox exposure without the need for viral infection with efficiencies 25- to 50-fold greater than those observed using direct infection and drug selection for pluripotency marker reactivation. We demonstrate that (i) various induction levels of the reprogramming factors can induce pluripotency, (ii) the duration of transgene activity directly correlates with reprogramming efficiency, (iii) cells from many somatic tissues can be reprogrammed and (iv) different cell types require different induction levels. This system facilitates the characterization of reprogramming and provides a tool for genetic or chemical screens to enhance reprogramming.

Directed reprogramming of somatic cells by defined factors provides a novel method for the generation of patient-specific stem cells with the potential to bypass both the practical and ethical concerns associated with somatic cell nuclear transfer (SCNT) and human embryonic stem (hES) cells. Although the generation of induced pluripotent stem (iPS) cells has proven a robust technology in mouse and human, a major impediment to the use of iPS cells for therapeutic purposes has been the viral-based delivery of the reprogramming factors because multiple proviral integrations pose the danger of insertional mutagenesis. Here we report a novel approach to reduce the number of viruses necessary to reprogram somatic cells by delivering reprogramming factors in a single virus using 2A “self-cleaving” peptides, which support efficient polycistronic expression from a single promoter. We find that up to four reprogramming factors (Oct4, Sox2, Klf4, and c-Myc) can be expressed from a single virus to generate iPS cells in both embryonic and adult somatic mouse cells and we show that a single proviral copy is sufficient to generate iPS cells from mouse embryonic fibroblasts. In addition we have generated human induced pluripotent stem (hiPS) cell lines from human keratinocytes, demonstrating that a single polycistronic virus can reprogram human somatic cells.

In Figure 4B of the above article, the first lane for measuring Zfp296 gene expression was inadvertently deleted when preparing the final figure; this caused a misalignment of this panel with the other panels in the figure. The modified figure below shows the corrected version for Figure 4B. We apologize for any inconvenience caused by this error.

Summary Elucidating how chromatin organization influences gene expression patterns and ultimately cell fate is fundamental to understanding development and disease. Here, we investigate the role of the histone variant H2AZ in embryonic stem (ES) cells because it plays an essential, but poorly understood function during development. Genome-wide analysis reveals that H2AZ is enriched at a large class of developmentally important genes whose promoters harbor bivalent histone modifications in a manner that is remarkably similar to the Polycomb group (PcG) protein Suz12. By using RNAi, we demonstrate a role for H2AZ in regulating target gene expression, find that H2AZ and PcG protein occupancy is interdependent at promoters, and further show that H2AZ is necessary for ES cell differentiation. Notably, H2AZ occupies a different subset of genes in lineage-committed cells suggesting that its dynamic redistribution is necessary for cell fate transitions. These results indicate that H2AZ, together with PcG proteins, may establish chromatin states necessary for the proper execution of developmental gene expression programs.